Non-splicing variants of gp350/220

ABSTRACT

Compositions comprising gp350 variant DNA and amino acid sequences are provided, as are vectors and host cells containing such sequences. Also provided is a process for producing homogeneous gp350 protein recombinantly and in the absence of production of gp220 protein, pharmaceutical compositions containing such protein and prophylactic treatments making use of such proteins.

This is a continuation of application Ser. No. 08/783,774, filed Jan.15, 1997 now U.S. Pat. No. 6,054,130; which is a continuation ofapplication Ser. No. 08/229,291, filed Apr. 18, 1994, now abandoned.

TECHNICAL FIELD

This invention relates to methods for making and using and compositionscontaining Epstein Barr virus (EBV) gp350 DNA and protein sequences.

BACKGROUND

Epstein-Barr virus (EBV), a member of the herpesvirus group, causesinfectious mononucleosis in humans. The disease affects more than 90% ofthe population. Health analysts estimate the cost of the disease in theUnited States is 100 million dollars per year. The virus is spreadprimarily by exchange of saliva from individuals who shed the virus.Children infected with EBV are largely asymptomatic or have very mildsymptoms, while adolescents and adults who become infected developtypical infectious mononucleosis, characterized by fever, pharyngitis,and adenopathy. People who have been infected maintain anti-EBVantibodies for the remainder of their lives, and are thus immune tofurther infection. Currently there is no commercially available EBVvaccine.

In addition to its infectious qualities, EBV has been shown to transformlymphocytes into rapidly dividing cells and has therefore beenimplicated in several different lymphomas, including Burkitt's lymphomaand oral hairy leukoplakia. EBV has also been detected in tissue samplesfrom nasopharyngeal tumors. Worldwide it is estimated that 80,000 casesof nasopharyngeal cancer occur and it is more prevalent in ethnicChinese populations.

Development of a live, attenuated vaccine for EBV has been and still isproblematic. Because of the potential oncogenic nature associated withEBV, researchers have been reluctant to use a live vaccine approach.This invention overcomes the problems associated with live vaccinedevelopment by creating methods and compositions for a subunit vaccine,that does not require the use of a potentially oncogenic live virus. Asubunit vaccine uses one or more antigenic proteins from the virus thatwill elicit an immune response and confer immunity.

Two of the more important antigenic EBV proteins are glycoprotein(s)gp350/300 and gp220/200 that form part of the viral membrane envelopeand allow virus particles to bind to and enter human target cells byinteracting with the cellular membrane protein, CD21. See Nemerow, J.Virology 61:1416(1987). They have long been singled out as subunitvaccine candidates but difficulties in obtaining antigenically activeprotein purified from native sources and low yields from recombinantlyproduced sources have hampered efforts of researcher and vaccinedevelopers. In the literature these proteins are referred to using avariety of molecular weight ranges (350 or 300 kilodaltons (kD) for oneof the proteins and 220 or 200 kDs for the other protein). The gp350 or300 protein is herein referred to as gp350 protein and the gp220 or 200protein is herein referred to as gp220 protein. Collectively, bothproteins are herein referred to as gp350/220 protein(s).

An alternatively spliced, single gene encodes the gp350/220 proteins andresults in the generation of gp350 and gp220 mRNA transcripts; nonaturally occurring variations in the gp350/220 gene splice sites areknown. The gene produces two expression products, the gp350 and gp220proteins. The open reading frame for the gp350/220 DNA sequence is 2721base pairs (bp). The entire reading frame encodes the 907 amino acids ofgp350. See U.S. Pat. No. 4,707,358 issued to Kieff (1987). The splicedversion of the reading frame covers 2130 bases and translates into gp220protein, a 710 amino acid sequence. The theoretical molecular weights ofgp350 protein and gp220 protein are 95 kD and 70 kD, respectively. Themeasured molecular weights of expressed gp350 protein and gp220 proteinvary but are approximately 350 kilodaltons and 220 kilodaltons (kD),respectively. The extensive glycosylation of the proteins accounts fordifference between the predicted and actual molecular weights. In anyone cell, both gp350 and gp220 proteins are produced at a molar ratioranging from about 6:1 to 1:1. For example, in B95-8 cells, which arepersistently infected with EBV, the ratio appears to vary but sometimesapproaches the 6:1 range. See, Miller, Proc. Natl. Acad. Sci.69:383(1972).

Similarly, recombinant production of these glycoproteins has heretoforeusually resulted in a mixture of gp350 and gp220 protein being produced.Heretodate, the gp350/220 proteins have been expressed in rat pituitary,Chinese hamster ovary VERO (African green monkey kidney) cells, as wellas in yeast cells. See, Whang, J. Virol. 61:1796(1982), Motz, Gene44:353(1986) and Emini, Virology 166:387(1988). A bovine papillomavirusvirus expression system has also been used to make gp350/220 proteins inmouse fibroblast cells. See, Madej, Vaccine 10:777(1992). Laboratory andvaccine strains of Vaccinia virus have also been used to express gp350/220 proteins. Modified recombinant versions of the EBV gp350/220 DNAand protein are known in the art. Specifically, recombinant truncatedconstructs of the gp350/220 gene lacking the membrane spanning sequencehave been made. Such constructs still produce a mixture of the two gp350 and gp220, but deletion of the membrane spanning region permitssecretion of the proteins. See, Finerty, J. Gen. Virology 73:449(1992)and Madej, Vaccine 10:777(1992). Also, various recombinantly producedrestriction fragments and fusion proteins comprising various gp350/220sequences have also been made and expressed in E. coli. See EP PatentPublication 0 173 254 published Jul. 24, 1991.

Accordingly, EBV research relating to gp350/220 heretodate has focusedeither on obtaining efficient expression of the native gp350/220sequence or on a modified sequence lacking the transmembrane domain,resulting in a mixture of the two alternate spliced versions of thenative or transmembrane lacking protein, or on production of epitopicfragment sequences in β-galactosidase fusion proteins.

Partially purified preparations of gp350/220 are known. See, Finerty, J.Gen. Virology 73:449(1992) (recombinantly produced, partially purified).With respect to native gp350/220 protein, in most instances, thepurification procedures resulted in inactivating the antigenicity of theprotein, making it unacceptable for use in a subunit vaccine. However,highly purified preparations of antigenically active gp350 protein fromnative (i.e., non-recombinant) sources have been reported in thescientific literature. See, David, J. Immunol. Methods 108:231(1988).Additionally recombinant vaccine virus expressing gp350/220 protein wasused to vaccinate cottontop tamarins against EBV-induced lymphoma. See,Morgan, J. Med. Virology 25:189(1988), Mackett, EMBO J. 4:3229(1985) andMackett, VACCINES '86, pp293(Lerner R A, Chanock R M, Brown F Eds.,1986, Cold Spring Harbor Laboratory). However, the viral gp350/220 DNAsequence has not heretofore been engineered so as to enable expressionsolely of either one of the alternate spliced versions of the gene,thereby enabling and ensuring the production of pure gp350 or gp220protein. Nor has a recombinant or mutant virus been made that expressesone or the other of the gp350 or gp220 proteins.

Generally, splice sites facilitate the processing of pre-mRNA moleculesinto mRNA. In polyoma virus, splice sites are required for the efficientaccumulation of late mRNA's. Alteration of the 3′ and 5′ splice sites inpolyoma virus transcripts decreased or completely blocked mRNAaccumulation. See, Treisman, Nature 292:595(1981). In SV40 virus,excisable intervening sequences facilitate mRNA transport out of thenucleus and mRNA stabilization in the nucleus and because theseintron/exon junction sequences facilitate binding of small, nuclear, RNPparticles, it is thought that prespliced mRNA's might fail to associateproperly with processing pathways. It has been shown that pointmutations at exon/intron splice sites reduce exon/intron cleavage andcan disrupt pre-mRNA processing, nuclear transport and stability. See,Ryu, J. Virology 63:4386(1989) and Gross, Nature 286:634(1980).

Therefore, until the present invention, the effect of splice sitemodification on the functional expression and antigenic activity of theproteins encoded by the EBV gp350/220 sequence was at best unknown andunpredictable.

Additional background literature includes the following. EBV biology anddisease is generally reviewed in Straus, Annal of Int. Med.118:45(1993). A description of the EBV BLLFI open reading frame is foundin Baer, Nature 310:207(1984). Descriptions of the Epstein-Barr virusgp350/220 DNA and amino acid sequences are found in articles by Beisel,J. Virology 54:665(1985) and Biggin, EMBO J. 3:1083(1984) and in U.S.Pat. No. 4,707,358 issued to Kieff, et al. (1987). A comparison of DNAsequences encoding gp350/220 in Epstein-Barr virus types A and B isdisclosed in Lees, Virology 195:578(1993). Monoclonal antibodies thatexhibit neutralizing activity against gp350/220 glycoprotein of EBV aredisclosed in Thorley-Lawson, Proc. Natl. Acad. Sci. 77:5307(1980).Lastly, splice site consensus sequences for donor and acceptor splicesites are disclosed in Mount, Nucleic Acids Res. 10:459(1982).

SUMMARY OF THE INVENTION

In one aspect this invention provides non-splicing variants of the EBVgp350/220 DNA sequence. The DNA sequences of the invention may includean isolated DNA sequence that encodes the expression of homogeneousgp350 protein. The DNA sequence coding for gp350 protein ischaracterized as comprising the same or substantially the samenucleotide sequence in FIG. 1 wherein the native nucleotides at thedonor and acceptor splice sites are replaced with non-nativenucleotides, and fragments thereof. This DNA sequence may include 5′ and3′ non-coding sequences flanking the coding sequence and further includean amino terminal signal sequence. FIG. 1 illustrates the non-codingsequences and indicates the end of the putative signal sequence with anasterisk. It is understood, however, that the DNA sequences of thisinvention may exclude some or all of these flanking or signal sequences.The non-splicing variant DNA sequences of the invention are produced byintroducing mutations into the FIG. 1 DNA sequence in the donor andacceptor splice sites of the gene encoding gp350/220. This eliminatesproduction of gp220 protein so that only the gp350 protein is produced.

Accordingly, in another aspect the invention comprises homogeneous gp350proteins, and methods of making the proteins by expression of thenon-splicing variant of EBV gp350/220 DNA sequence in an appropriateprokaryotic or eukaryotic host cell under the control of suitableexpression control sequence. As the term is used here with respect togp350 proteins, homogeneous means free or substantially free from gp220protein. We note that homogeneous gp350 protein, recombinantly producedin mammalian or insect cells, has not to our knowledge ever beenreported in the scientific literature heretofore.

In yet another aspect, homogeneous gp350 proteins, additionally havingdeletions resulting in a secreted product are provided. Such deletionscomprise either removal of the transmembrane region or removal of thetransmembrane region and the remaining C-terminus of gp350. Suchadditionally modified DNA sequences and the proteins encoded thereby areyet another aspect of this invention.

Also provided is a recombinant DNA molecule comprising vector DNA and aDNA sequence encoding homogeneous gp350 protein. The DNA moleculeprovides the gp350 sequence in operative association with a suitableregulatory sequence capable of directing the replication and expressionof homogeneous gp350 in a selected host cell. Host cells transformedwith such DNA molecules for use in expressing recombinant homogeneousgp350 are also provided by this invention.

The DNA molecules and transformed host cells of the invention areemployed in another aspect of the invention, a novel process forproducing recombinant homogeneous gp350 protein or fragments thereof. Inthis process a cell line transformed with a DNA sequence encoding ahomogeneous gp350 protein or fragment thereof (or a recombinant DNAmolecule as described above) in operative association with a suitableregulatory or expression control sequence capable of controllingexpression of the protein is cultured under appropriate conditionspermitting expression of the recombinant DNA. The expressed protein isthen harvested from the host cell or culture medium by suitableconventional means. The process may employ a number of known cells ashost cells; presently preferred are mammalian cells and insect cells.

The DNA sequences and proteins of the present invention are useful inthe production of therapeutic and immunogenic compounds having EBVantigenic determinants. Such compounds find use in subunit vaccines forthe prophylactic treatment and prevention of EBV related diseases, suchas mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma.Accordingly, in yet another aspect the invention comprises suchtherapeutic and/or immunogenic pharmaceutical compositions forpreventing and treating EBV related conditions and diseases in humanssuch as infectitious mononucleosis, Burkett's lymphoma andnasopharyngeal carcinoma. Such therapeutic and/or immunogenicpharmaceutical compositions comprise a immunogenically inducingeffective amount of one or more of the homogeneous gp350 proteins of thepresent invention in admixture with a pharmaceutically acceptablecarrier such as aluminum hydroxide, saline and phosphate buffered salineas are known in the art. By “immunogenically inducing” we mean an amountsufficient for stimulating in a mammal the production of antibodies toEBV. Alternatively, the active ingredient may be administered in theform of a liposome-containing aggregate. For prophylactic use, suchpharmaceutical compositions may be formulated as subunit vaccines foradministration in human patients. Patients may be vaccinated with a dosesufficient to stimulate antibody formation in the patient; andrevaccinated after six months or one year.

A further aspect of the invention therefore is a method of treating EBVrelated diseases and conditions by administering to a patient,particularly to a human patient, an immunogenically inducingtherapeutically effective amount of a homogeneous gp350 protein in asuitable pharmaceutical carrier. Still another aspect of the inventionis a method of stimulating an immune response against EBV byadministering to a patient an immunogenically inducing effective amountof a homogeneous gp350 protein in a suitable pharmaceutical vehicle.

Other aspects and advantages of the invention are described further inthe following detailed description.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1D illustrates the DNA and amino acid sequence of gp350/220(From Beisel, J. Virology 54:665(1985)). The donor and acceptor splicesites are indicated. The transmembrane region is delineated with thehorizontal arrows and an asterisk (*) marks the end of the putativesignal sequence. Nucleotide numbering is shown at the left; amino acidnumbering at the right.

FIGS. 2A-2B illustrates construction of gp350 deletion and site directedmutants. The plasmid maps labelled pMDTM and pMSTOP exemplify thenon-splicing gp350/220 variants of the invention. In section (A), alinear model of the gp350 protein is shown approximately to scale withthe encoding clone, BLLF1, below. An N-terminal signal sequence (SS) andthe transmembrane domains (TM) are indicated on the protein andimportant restriction sites are indicated on the gene diagram. The gp350gene was cloned in two segments, the HindIII/BfaI BLSH1 fragment and theBanI/HindIII BLSH2 fragment. SCYT was created using the polymerase chainreaction from the region of BLLF1 indicated. In (B), the cloning schemefor pDTM, pSTOP, pMDTM, and pMSTOP is illustrated (plasmids not toscale). The details of the cloning are described in Examples 1 and 2.Plasmid maps are marked with the relevant restriction sites, the cloningvectors used and the gp350 gene fragments. Splice site mutations inpMDTM and pMSTOP are indicated by asterisks.

FIG. 3 illustrates the results of immunoprecipitation of homogeneousgp350 protein from pMDTM clones as analyzed by SDS-PAGE. Positivecontrol (GH3Δ19) cells secreting a truncated form of the gp350/220proteins, negative control (pEE14) cells and several pMDTM clones weremetabolically labeled with ³⁵S-methionine for 5.5 hours; homogeneousgp350 protein was immunoprecipitated from the resulting tissue culturesupernatants. For each cell type, samples of labeled tissue culturesupernatants (S) and gp350/220 precipitations (Ip) were electrophoresedon 5% SDS-PAGE (polyacrylamide gel electrophoresis). Location ofmolecular weight markers are indicated on the left side.

DETAILED DESCRIPTION

Disclosed are compositions and methods comprising cloned EBV DNAsequences encoding non-splicing variants of gp350 protein. As noted,such non-splicing variants are referred to herein as homogeneous gp350proteins. Normally, when the gp350/220 gene is expressed in mammaliancells two gene products are generated, gp350 and gp220, due to RNAsplicing of the gene. The invention allows for only one gene product,gp350, to be produced. The invention involves removing some or all ofthe RNA splice site signals in the gp350 gene and expressing the gene ina suitable host cell. Mutations in the gp350/220 gene were introduced toprevent production of the 220 kD version of the protein when thegp350/220 gene is expressed in mammalian cells. As a result, mRNAtranscripts encoding only gp350 are produced. The elimination of gp220expression by using a gp350/220 gene non-splicing variant will result inincreased production of gp350 relative to gp220. Production of gp220 isnot essential for production of an effective anti-EBV vaccine becausegp350 contains all the potential antigenic sites found on gp220.

Therefore, one aspect of this invention provides a DNA sequence encodinga polypeptide sequence substantially the same as gp350, except that thedonor splice site codon encoding amino acid 501 and the acceptor splicesite codon encoding amino acid 698 have been modified by replacement ofnative nucleotides with non-native nucleotides. Preferably the nativenucleotides are replaced with non-native nucleotides such that the aminoacid sequence remains the same. Specifically, in the example, nativenucleotides AAGT at the donor splice site (nucleotides 1500 through1504) and native nucleotides A and T flanking the GG acceptor splicesite (nucleotides 2091 and 2094) were replaced with nucleotides GTCA andT and A, respectively. Consequently, the Glutamine at amino acidposition 500 and the Serine at position 501 remained the same as aresult of this substitution in the donor site. Likewise, the Threonineat amino acid position 697 and the Glycine at position 698 remained thesame as a result of the modification in the acceptor site.

Analogously, substitutions other than those specifically exemplifiedcould readily be performed by one skilled in the art as is more fullydescribed below.

Therefore, in one aspect the invention comprises homogeneous gp350proteins. The homogeneous gp350 proteins are further characterized byhaving an amino acid sequence substantially the same as that shown inFIG. 1 from amino acids 1 through 907, from amino acids 1 through 862 orfrom amino acids 1 through 907 and excepting amino acids 863 through881, each with or without the N-terminal 18 amino acid signal sequence.In addition, analogs of homogeneous gp350 proteins are provided andinclude mutants in which there are variations in the amino acidssequence that retain antigenic activity and preferably have a homologyof at least 80%, more preferably 90%, and most preferably 95%, with thecorresponding region of the homogeneous gp350 proteins. Examples includeproteins and polypeptides with minor amino acid variations from theamino acid sequence of FIG. 1; in particular, conservative amino acidsreplacements. Conservative replacements are those that take place withina family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into fourfamilies. (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan and tyrosine are sometimes classified jointlyas aromatic amino acids. For example, it is reasonable to expect that anisolated replacement of a leucine or a similar conservative replacementof an amino acid with a structurally related amino acid will not have amajor effect on antigenic activity or functionality.

The invention offers the advantage of simpler purification of gp350.Because gp350 and gp220 have similar biochemical properties, gp220 isoften co-purified in preparations of gp350. Cells expressing only thenon-splicing variant of the gp350/220 gene simplifies proteinpurification. This will reduce the costs of producing gp350. Theinvention also makes biochemical characterization of the startingmaterial for gp350 purification easier. Because only one species ispresent, protein content analysis and amino acid sequence analysis maybe performed without accounting for the presence of a second species.

The invention additionally offers the advantage of increased gp350production. Prevention of gp350 gene splicing will shift the cell fromdual production of gp350 and gp220 to the production of gp350 alone. Insome cells, the concentrations of gp220 have been estimated to be30%-100% of the gp350 concentration. With the gene splicing eliminated,gp350 production will be increased by the lack of gp220 production.

The DNA sequence of the gp350/220 gene is described by Beisel, J.Virology 54:665(1985) and Biggin, EMBO J. 3:1083(1984) and isillustrated in FIG. 1. The gene is an open reading frame of 2721 bases,encoding 907 amino acids and specifying a primary translation product ofabout 95 kD. The difference between predicted and actual valuesrepresents extensive glycosylation of the protein. 591 bases (encoding197 amino acids) are spliced out to produce gp220. The apparentmolecular weight of gp350/220 gene products may also vary depending uponthe type of measurement system used, glycosylation site utilization indifferent cell types, post-translational processing differences orselective gene mutation. Measured values vary for the products ofdifferent gp350/220 gene non-splice site variants but the term“homogeneous gp350 protein or proteins” encompasses gene products of thenon-splicing variant, optionally having additional deletions ormutations such as the C-terminal deletions and/or transmembranemodifications also disclosed herein. The term “gp220 protein” refers tothe alternatively spliced gp350/220 gene product with a molecular weightof approximately 220 kD. Splice-sites in the gp350/220 gene wereidentified by comparison of the gp350/220 gene with consensus donor andacceptor splice sequences based on other genes, predominantly fromeukaryotic organisms. The consensus sequences developed by Mount,Nucleic Acids Res. 10:459(1982) from studying the splice sites in othergenes are:

Donor: A/C AG/G*T*A/G AGT

Acceptor: T/C N₁₁C/TA*G*/G

The bases asterisked above represent bases that appear in 100% of allsplice sites (highly conserved). Positions with two bases or one baserepresent conserved positions (non highlighted positions). The slashindicates the actual site of splicing.

In the gp350/220 gene the donor splice site occurs after nucleotide 1501and the acceptor splice occurs after nucleotide 2092, as shown by DNAsequencing (Biggin. EMBO J. 3:1083(1984)) of the gp350/220 gene. (Thenumbering used herein and in FIG. 1 conforms to the numbering inBiggin). The splice site occurs in the corresponding gene region in theType B strain of EBV (the donor splice site after A₁₅₀₁ and the acceptorsplice site after G₂ ₂₀₂₉). The invention encompasses compositions madeusing either the A or B strain or another EBV strain's splice site toproduce a single species of mRNA from the gp350/220 gene. The DNAsequence of the Type A form of the virus from strain B95-8 was used inthe Examples although the DNA sequence of the Type B strain couldequally have been used, because the translated gene products of Type Aand B strains are 98% identical. The B strain lacks amino acids 507through 520 and 570 through 576. The type A strain was used because itcontains all the possible gp350 antigenic sites. Alternatively, EBVgp350/220 having strain-specific sequences could be used in accordancewith the teachings herein to produce EBV strain-specific homogeneousgp350 proteins having immunogenic properties specific to a particularstrain and therefore useful in immunogenic and/or therapeuticcompositions for the prevention or treatment of strain specific EBVrelated diseases. Table 1 shows the wild type nucleotide and amino acidsequences of the donor and acceptor splice sites.

To prevent RNA splicing of the gp350/220 gene, mutations were introducedinto the gp350/220 gene nucleic acid sequence to replace the relevantbase pairs of the RNA splice site. To render a splice-sitenonfunctional, preferably at least one of the bases out of the twohighly conserved bases framing the donor site or acceptor site should bereplaced with nonconserved bases, more preferably at least two highlyconserved bases should be mutated to nonconserved bases. Other conservedbases, more than two bases away from the splice site, can also bereplaced with nonconserved splice site bases to further decreaserecognition of the splice site. Both the donor and the acceptor site canbe changed to impair splicing mechanisms. Preferably, both the donor andthe acceptor contain at least one change each, in one of the four highlyconserved splice site base positions, and more preferably at least twochanges in two of the four highly conserved splice site base positions.If one splice site is not mutable due to a desire to maintain thewild-type amino acid sequence then it is preferable to introduce atleast two mutations to the other splice site.

Mutation at the gp350/220 splice sites may introduce changes into theamino acid sequence of the subsequently expressed gp350 protein.Preferably such changes should be conservative amino acid substitutions.Conservative substitutions in the amino acid sequence, as opposed tononconservative changes in the amino acid sequence, will help preserveantigenic sites. Conservative amino acid changes can be made as long asthe base change (or base changes) result in a suitable change in theinvariant donor/acceptor bases. For example, Gly could be substitutedfor Ser₅₀₁ at the donor splice site, using any Gly-specific codons otherthan GGU (use of GGU would preserve the G nucleotide and would notresult in the desired GT replacement in the splice signal). Likewise, atthe acceptor splice site, Gly₆₉₈ to Ala would be a conservative change,but since all Ala codons start with the highly conserved G nucleotide,this would not result in the desired replacement. Although Proline alsomight be a conservative amino acid change, proline would not be used toreplace a wild type amino acid because it would result in modificationof the tertiary structure of the protein and thereby mask one or moregp350 antigenic sites. Table 1 shows the acceptable conservative aminoacid replacements in the wild-type sequences. At the bottom of Table 1is an example of a mutation with conservative amino acid changes.

TABLE 1 Donor Acceptor Wild-type Sequences splice splice GAA A | GT ACAG | GT Glu Ser₅₀₁ Thr Gly₆₉₈ ↓ ↓ ↓ ↓ Conservative a.a. changes Asn AlaAla Ser Asp Gly Gly Thr Gln Thr Ser ex.: GAC ACA TCG TCT Asp Thr₅₀₁ SerSer₆₉₈

Although one aspect of the present invention comprises a non-splicingvariant of gp350/220, additional mutations of the gp350/220 codingsequence may also be desirable. In order to produce soluble homogeneousgp350 proteins (“soluble proteins” are either free in solution ormembrane associated but are not membrane integrated), for example, toavoid cell toxicity problems incurred by the expression of full lengthgp350 as an integral membrane protein, the membrane spanning region(also known as the transmembrane region) of gp350 is modified bydeletion of all or part of its encoding DNA sequence. The membranespanning region of gp350/220 comprises amino acids 861 (methionine)through 881 (alanine). See, Beisel, J. Virology 54:665(1985).Preferably, at least 8 amino acids of the transmembrane region aredeleted, more preferably at least 12 amino acids are deleted and mostpreferably between 18 and 21 amino acids are deleted. Accordingly, inanother aspect, the invention provides non-splicing variants ofgp350/220 DNA and/or gp350 homogeneous protein additionally comprisingat least one deletion in the transmembrane region of the gp350/220 DNAand/or gp350 homogeneous protein that results in the expression ofsoluble homogeneous gp350 protein.

In addition to deleting all or part of the transmembrane domain of thenon-splicing gp350/220 variant, the C-terminal sequence following thetransmembrane domain and comprising amino acids 881 through 907 may alsobe deleted in whole or in part, as described herein, in accordance withthe invention. Thus, in another aspect the invention comprisesnon-splicing variants of gp350/220 DNA and/or homogeneous proteinfurther modified by deletion of all or a portion of the DNA encodingand/or amino acid sequence comprising the transmembrane region ofgp350/220 and even further modified by deletion of the remainingC-terminal DNA and/or amino acid sequences of gp350/220.

Accordingly, in another aspect the invention comprises non-splicingvariant DNA sequences encoding the homogeneous gp350 proteins of theinvention. Such DNA sequences comprise the DNA sequence of FIG. 1encoding amino acids 1 through 907 and further comprising the nucleotidesubstitutions taught herein to remove the donor and acceptor splicesites. Such DNA sequences optionally comprise truncated DNA sequences inwhich the nucleotides encoding all or part of the transmembrane domainand C-terminus comprising amino acids 861 through 907 are deleted anddeletion variants in which the nucleotides encoding all or part of thetransmembrane domain comprising amino acids 861 through 881 are deleted.The DNA sequences of the present invention encoding homogeneous gp350proteins may also comprise DNA capable of hybridizing under appropriatestringency conditions, or which would be capable of hybridizing undersuch conditions but for the degeneracy of the genetic code, to anisolated DNA sequence of FIG. 1. Accordingly, the DNA sequences of thisinvention may contain modifications in the non-coding sequences, signalsequences or coding sequences, based on allelic variation, speciesvariation or deliberate modification.

These non-splicing variant gp350/220 DNA sequences as disclosed hereincan be constructed using methods well known in the art. The modified DNAsequences of this invention can be expressed recombinantly, likewiseusing known methods, to produce the homogeneous gp350 proteins of thisinvention. Such recombinant proteins can be purified and incorporatedinto pharmaceutical compositions for the prophylactic treatment andprevention of EBV related diseases.

The non-splicing variants of gp350/220 DNA of this invention can beexpressed recombinantly in different types of cells using theappropriate expression control systems as is known in the art. Suitablecells known and available in the art include, but are not limited to,yeast cells such as Saccharomyces cerevisiae, bacterial cells such as E.coli and Bacillus subtilis and mammalian cells such as GH3, CHO, NSO,MDCK and C-127 cells. Vectors used with cell types are selected based ontheir compatibility with the cell type and expression control systemused. Cells and vectors that allow for the expression of secretedproducts of the gp350/220 gene are preferred. Typically for example, E.coli is transformed using derivatives of pBR322 which have been modifiedusing conventional techniques to contain the DNA sequences forexpression of the desired protein, in this instance the non-splicingvariant sequences of EBV gp350, with or without the sequences encodingthe C-terminus and/or membrane spanning region. pBR322 contains genesfor ampicillin and tetracycline resistance, which can be used asmarkers. See, Bolivar, Gene 2:95(1977). Commonly used expression controlsequences, i.e., promoters for transcription initiation and optionallyan operator or enhancer, include the beta-lactamase and lac promotersystems (see Chang, Nature 198:1056(1977)), the tryptophan promotersystem (see Goeddel, Nucleic Acids Res. 8:4057(1980)) and thelambda-derived PL promoter and N-gene ribosome binding site (seeShimatake, Nature 292:128(1981). However, any available promoter systemor expression control system that is compatible with prokaryotic hostcells can be used. Other exemplary host cells, plasmid and expressionvehicles are disclosed in U.S. Pat. No. 4,356,270 issued to Itakura(1982), U.S. Pat. No. 4,431,739 issued to Riggs (1984) and U.S. Pat. No.4,440,859 issued to Rutter (1984).

Insect cells may also be used as host cells employing insect cellexpression. In the case of expression in insect cells, generally thecomponents of the expression system include a transfer vector, usually abacterial plasmid, which contains both a fragment of the baculovirusgenome, and a convenient restriction site for insertion of theheterologous gene or genes to be expressed; a wild type baculovirus witha sequence homologous to the baculovirus-specific fragment in thetransfer vector (this allows for the homologous recombination of theheterologous gene in to the baculovirus genome); and appropriate insecthost cells and growth media.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a procaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus transfer vector can alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression can be either regulated orconstitutive. For insect cell expression technology, see EP patentpublication 155 476.

Yeast, for example Saccharomyces cervisiae, may also be used as a hostcell. Various strains are available and may be used. Likewise, plasmidvectors suitable for yeast expression are known, as are promoter andexpression control systems. See for example, Myanohara, Proc. natl.Acad. Sci. 80:1(1983)(PHO5 promoter), EP Patent Publication 012 873(leader sequences), Kurtz, Mol. Cell. Biol. 6:142(1986), Ito, J.Bacteriol. 153:163(1983) and Hinnen, Proc. Natl. Acad. Sci. 75:1929(1979)(transformation procedures and suitable vectors).

Eukaryotic cells from multicellular organisms may of course also be usedas hosts cells for the expression of genes encoding proteins andpolypeptides of interest. Useful host cell lines include VERO and HeLacells, and Chinese hamster ovary cells (CHO). Expression vectorscompatible with such cells are also available and typically includepromoters and expression control sequences, such as for example, theearly and late promoters from SV40 (see Fiers, Nature 273:113(1978)) andpromoters from polyoma virus, adenovirus 2, bovine papilloma virus oravian sarcoma virus. Exemplary host cells, promoters, selectable markersand techniques are also disclosed in U.S. Pat. No. 5,122,469 issued toMather (1992), U.S. Pat. No. 4,399,216 issued to Axel (1983), U.S. Pat.No. 4,634,665 issued to Axel (1987), U.S. Pat. No. 4,713,339 issued toLevinson (1987), U.S. Pat. No. 4,656,134 issued to Ringold (1987), U.S.Pat. No. 4,822,736 issued to Kellems (1989) and U.S. Pat. No. 4,874,702issued to Fiers (1989).

Transformation of suitable host cells is accomplished using standardtechniques appropriate to such cells, such as CaCl₂ treatment forprokaryotes as disclosed in Cohen Proc. Natl. Acad. Sci. 69:2110(1972)and CaPO₄ precipitation for mammalian cells as disclosed in Graham,Virology 52:546(1978). Yeast transformation can be carried out asdescribed in Hsiao, Proc. Natl. Acad. Sci. 76:3829(1979) or as describedin Klebe, Gene 25:333(1983).

The construction of suitable vectors containing the non-splicing variantgp350 sequence (with or without the additional modifications disclosedhere resulting in deletion of the C-terminus and/or the membranespanning region) is accomplished using conventional ligation andrestriction techniques now well known in the art. Site specific DNAcleavage is performed by treating with suitable restriction enzyme(s)under standard conditions, the particulars of which are typicallyspecified by the restriction enzyme manufacturer. Polyacrylamide gel oragarose gel electrophoresis may be performed to size separate thecleaved fragments using standard techniques and the fragments bluntended by treatment with the Klenow fragment of E. coli polymerase I inthe presence of the four deoxynucleotide triphosphates. Treatment withS1 nuclease hydrolyzes any single-stranded portions. Syntheticoligonucleotides can be made using for example, thediethylphosphoamidite method known in the art. See U.S. Pat. No.4,415,732 (1983). Ligations can be performed using T4 DNA ligase understandard conditions and temperatures and correct ligations confirmed bytransforming E. coli or COS cells with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers as are known in the art.

Such recombinant DNA techniques are fully explained in the literature.See, e.g. Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED.(1989); DNA CLONING, Vol. I and II (D N Glover ed 1985); OLIGONUCLEOTIDESYNTHESIS (M J Gait ed 1984); NUCLEIC ACID HYBRIDIZATION (B D Hames ed1984); TRANSCRIPTION AND TRANSLATION (B D Hames ed 1984); ANIMAL CELLCULTURE (R I Freshney ed 1986); B. Perbal, A PRACTICAL GUIDE TOMOLECULAR CLONING (1984); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J HMiller ed 1987 Cold Spring Harbor Laboratory); Scopes, PROTEINPURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed, (1987 Springer-Verlag NY)and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY Vols I-IV (D M Weired 1986). Allsuch publications mentioned herein are incorporated by reference for thesubstance of what they disclose.

Accordingly in another aspect the invention comprises vectors containingthe non-splicing variants of gp350/220 DNA sequences and host cells andfurther comprises a method of making a non-splicing variant of gp350/220protein by culturing said host cells containing a vector that iscarrying a non-splicing variant of a gp350/220 DNA sequence operativelylinked to an expression control sequence under culture conditionsenabling expression of the homogeneous gp350 protein.

The expressed homogeneous gp350 is purified from cell and culture mediumconstituents using conventional glycoprotein purification techniquessuch as, but not limited to, ultrafiltration, free flow electrophoresis,gel filtration chromatography, affinity chromatography, SDS-PAGE,differential NH₄SO₄ precipitation, lectin columns, ion exchange columnsand hydrophobicity columns as is known in the art. Small scaleanalytical preparations of gp350 are most readily purified usingSDS-PAGE or lectin affinity columns and such small scale preparationsfor use in vaccination or immune response experiments are most readilypurified using liquid chromatography. For large scale production ofcommercially significant quantities of gp350 for use in vaccinecompositions, a combination of ultrafiltration, gel filtration, ionexchange, and hydrophobic interaction chromatography are preferred.

The purified, homogeneous gp350 proteins of the present invention may beemployed in therapeutic and/or immunogenic compositions for preventingand treating EBV related conditions and diseases such as infectitiousmononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma. Suchpharmaceutical compositions comprise an immunogenically-inducingeffective amount of one or more of the homogeneous gp350 proteins of thepresent invention in admixture with a pharmaceutically acceptablecarrier, for example an adjuvant/antigen presentation system such asalum. Other adjuvant/antigen presentation systems, for instance, MF59(Chiron Corp.), QS-21 (Cambridge Biotech Corp.), 3-DMPL(3-Deacyl-Monophosphoryl Lipid A) (RibiImmunoChem Research, Inc.),clinical grade incomplete Freund's adjuvant (IFA), fusogenic liposomes,water soluble polymers or Iscoms (Immune stimulating complexes) may alsobe used. Other exemplary pharmaceutically acceptable carriers orsolutions are aluminum hydroxide, saline and phosphate buffered saline.The composition can be systemically administered, preferablysubcutaneously or intramuscularly, in the form of an acceptablesubcutaneous or intramuscular solution. Also inoculation can be effectedby surface scarification or by inoculation of a body cavity. Thepreparation of such solutions, having due regard to pH, isotonicity,stability and the like is within the skill in the art. The dosageregimen will be determined by the attending physician consideringvarious factors known to modify the action of drugs such as for example,physical condition, body weight, sex, diet, severity of the condition,time of administration and other clinical factors. Exemplary dosageranges comprise between about 1 μg to about 1000 μg of protein.

In practicing the method of treatment of this invention, animmunologically-inducing effective amount of homogeneous gp350 proteinis administered to a human patient in need of therapeutic orprophylactic treatment. An immunologically inducing effective amount ofa composition of this invention is contemplated to be in the range ofabout 1 microgram to about 1 milligram per dose administered. The numberof doses administered may vary, depending on the above mentionedfactors. The invention is further described in the following examples,which are intended to illustrate the invention without limiting itsscope.

EXAMPLE 1 Deletion of the gp350/220 Transmembrane Region andTransmembrane Region through C-terminus to create pDTM and pSTOP

The gp350/220 gene from the EBV B95-8 strain (Miller, et al., 1972), isavailable in a BamHI library as an open reading frame called BLLF1(Baer, Nature 310:207, 1984). To create the desired constructs (showndiagrammatically in FIG. 2B), the gp350/220 gene was cloned in twoparts: 1) BLSH1, a 2.3 kb HindIII/BfaI 3′ fragment and 2) BLSH2, a 337b.p. BanI/HindIII 5′ fragment (FIG. 2A). These fragments were clonedinto staging vectors so that the deletions of the C-terminal cytoplasmicand/or transmembrane-encoding domains could be performed. Because theBfaI site occurs at the 5′ end of the region encoding the gp350transmembrane (TM) domain, it was used to construct the TM domaindeletions and TM domain deletions with adjacent C-terminus deletions.Using BfaI, it was possible to create deletions retaining only two aminoacids of the TM region (Table 2).

1. Construction of pDTM From pSTG1, and pSTG3

The plasmid pDTM is comprised of a gp350/220 nucleic acid sequence thatlacks a complete TM coding region. This construct was made using twostaging vectors pSTG1 and pSTG3. A 450 bp PCR product, SYCT, thatintroduced a BfaI site at the 3′ end of the TM region was made using aBLLF1 clone target sequence (FIG. 2). The PCR primers used are asfollows:

       BfaI Primer 1: GG ATC CTA GAC TGC GCC TTT AGG CGT A BLLF1:       ... GAC TGC GCC TTT AGG CGT A.. A.A.:        ... Asp Cys Ala PheArg Arg ...           |_(———)End of TM Region Primer 2: GGA TCC TCT GTTCCT TCT GCT CCA GTG BLLF1: ... ... TCT GTT CCT TCT GCT CCA GTG

The BfaI site of Primer 1 was used to clone a BfaI/XmaI fragment of SCYTinto pSTG1. The remainder of Primer 1 corresponds to the amino acidsequence encoded by clone BLLF1. Primer 2 corresponds to a regionoutside the gp350/220 open reading frame on the 3′ side of the gene. TheSCYT PCR fragment was cut with BfaI and XmaI to produce a 136 base pairfragment which was cloned into a pMT11 vector (Spaete and Mocarski,1985) along with a second fragment, a BLSH1 HindIII/BfaI fragment, tocreate pSTG1. Sequencing across the BfaI site indicated that all of theTM amino acid coding region was deleted except for amino acids Met andLeu (see Table 2). A third BLLF1 fragment, BLSH2, was cloned into pMT11to create pSTG3. A 16 base pair BanI/XbaI oligonucleotide linker outsideof the gp350/220 gene coding sequence was used to clone the BLSH2BanI/HindIII fragment into the pSTG3. A 2.4 HindIII/XmaI pSTG1 fragment,was cloned into a pEE14 vector (Celltech, England) together with a 0.3XbaI/HindIII pSTG3 fragment to complete the pDTM construct.

2. Construction of pSTOP using Vectors pSTG2 and pSTG3

The plasmid pSTOP comprises a gp350/220 gene that lacks a TM region andthe C-terminal cytoplasmic region adjacent the TM region. To create thisconstruct, a 16 base pair BfaI/EcoRI oligonucleotide linker was createdwith stop codons (underlined) in three frames following the BfaI stickyend as shown below:

TAT AGA CTA GTC TAGG A TCT GAT CAG ATC CTT AA

The 5′ overhang (TA) of the upper sequence is a sticky end for a BfaIrestriction site and the 5′ overhang (TTAA) of the lower sequence is anEcoRI sticky end. This 16 base pair linker was used to clone a BLSH1HindIII/BfaI fragment into pMT11, in order to create pSTG2. A 2.3 kbpSTG2 HindIII/EcoRI fragment and the pSTG3 0.3 kb XbaI/HindIII fragmentwere cloned into pEE14 to create pSTOP.

3. Comparison of the Wild-type, pDTM and pSTOP Sequences at the TMRegion

The oligonucleotide sequence and translated amino acid sequence of thewild type, pSTOP, and pDTM 3′ ends of gp350 DNA and amino acid sequencesare shown in Table 2 below. Arrows indicate the beginning and end of thewild-type transmembrane domain (TM). Only two amino acids from thetransmembrane domain are retained in pDTM and pSTOP, Met₈₆₁ and Leu₈₆₂(see also FIG. 1). Note that a stop codon immediately follows Leu₈₆₂ inpSTOP. In pDTM the former location of the deleted transmembrane regionis marked “ΔTM”. (In the Table, the native amino acids are indicated.)

TABLE 2 3′ End of gp350 Wild-Type Sequence ...AAC CTC TCC ATG CTA GTACTG.....GTC ATG GCG GAC TGC GCC ...Asn Leu Ser Met Leu₈₆₂ ValLeu.....Val Met Ala Asp₈₈₂ Cys Ala              ↑                                  ↑             TMstart                          TM end 3′ End of pSTOP ...AAC CTC TCC ATGCTA TAG ACT AGT TCT AGG ... ...Asn Leu Ser Met Leu₈₆₂ STOP 3′ End ofpDTM ...AAC CTC TCC ATG CTA GAC TGC GCC... ...Asn Leu Ser Met Leu₈₆₂Asp₈₈₂ Cys Ala....                         Λ                          ΔTM

EXAMPLE 2 Removal of the gp3501220 Gene Donor and Acceptor Splice Sitesto Create pMDTM and pMSTOP

In order to obtain homogeneous production of a gp350 protein the highlyconserved and conserved bases of the gp350/220 gene splice site werechanged. Four bases were changed in the donor splice site, including thehighly conserved GT pair that occurs in 100% of all splice sites. Twoconserved donor site bases, AA, were replaced with GT. The two highlyconserved (invariant) donor splice site bases were changed from GT toCA. At the acceptor splice site, only one of the highly conservedacceptor splice site bases was altered to preserve the amino acidsequence. A second conserved acceptor splice site base was changed asindicated in Table 3. Table 3 summarizes the bases changed in the donorand acceptor splice sites of the gp350/220 gene.

TABLE 3 EBV gp350/220 Gene Splice Site Changes Donor Splice site: donordonor Wild-type: GAA A↓GT mutant: GAG* T*C*A* Glu Ser₅₀₁ Glu Ser₅₀₁Acceptor Splice site: acceptor acceptor Wild-type: ACA G↓GT mutant: ACT*GGA* Thr Gly₆₉₈ Thr Gly₆₉₈

The bases changed by oligonucleotide-based mutagenesis are marked withan asterisk in the mutant sequences. The actual site of splicing isindicated by an arrow, and the encoded amino acids are shown. Note thatthe amino acid sequence does not change as a result of the nucleotidesubstitutions.

These nucleotide substitutions to the wild type gp350/220 donor splicesite and accepter splice site DNA sequences were accomplished usingoligonucleotide-mediated mutagenesis. A modified phage vector, M13TAC,was employed to produce mutations as described in Zoller, M. E. andSmith, M. (1983) Methods of Enzymol. 100:468. BamHI/XhoI fragments ofthe gp350/220 nucleotide sequence were cloned into the polylinker ofplasmid M13TAC using Asp718 and BamHI restriction sites on thepolylinker, combined with a 19 bp oligonucleotide Linker containingAsp718 and XhoI sticky ends. The plasmids M13DTM and M13STOP of Example1 (FIG. 2B), were used for the mutagenesis.

Two 42-mer oligonucleotides, PrDonorl and PrAcceptorl, were made for usein the mutagenesis. Each was designed to be complementary to gp350/220gene sequences centering on either the donor or acceptor splice sites.The only region of the oligonucleotides that were not complementary tothe gp350/220 gene were the bases representing the desired mutations.Mutagenesis oligonucleotides comprised the following:

PrDonor1 Primer: GGT CAT GTC GGG GGC CTT TG ¦A  CTC TGT GCC GTT GTC CCATGG                         ** ¦*  * EBV: GGT CAT GTC GGG GGC CTTAC ¦T  TTC TGT GCC GTT GTC CCA TGG PrAcceptor1 Primer: CTG TGT TAT ATTTTC ACC TC ¦C  AGT TGG GTG AGC GGA GGT TAG                         *  ¦  * EBV: CTG TGT TAT ATT TTC ACC AC ¦C  TGT TGG GTG AGC GGA GGT TAG

The sequence of the mutagenesis oligonucleotides are labelled “Primer,”while the DNA sequence spanning the gp350/220 gene splice sites arelabelled “EBV.” Bases that were changed as a result of the mutagenesisare marked with an asterisk. The dashed line indicated the location ofthe splice.

The oligonucleotides PrDonor1 and PrAcceptor1 were hybridized tosingle-stranded clones of M13-DTM and M13-STOP. T4 DNA polymeraseholoenzyme was used to produce double-stranded M13 DNA and E. coli wastransformed with the double-stranded DNA. Using the vector M13TAC, anyclone that contained the desired mutation could be identified by a colorchange from white to blue in the presence of X-gal andisothiopropylgalactate. Blue plaques were picked and grown up, and DNAsequencing across splice junctions was used for the final identificationof mutant clones, labelled M13-MDTM and M13-MSTOP.

After identifying clones containing the desired mutations, BamHI/XhoIfragments were cut out of M13-MDTM and M13-MSTOP and ligated back intopDTM or pSTOP backbones to create the constructs pMDTM and pMSTOP,respectively. These constructs were transfected into CHO cells toexpress the non-splicing variant gp3501220 DNA sequences as described inExample 3.

EXAMPLE 3 Expression of gp350 in CHO Cells

1. Transfection of gp350/220 Gene Constructs

One method for producing high levels of homogeneous gp350 protein of theinvention from mammalian cells involves the construction of cellscontaining multiple copies of the heterologous gp350 DNA sequence. Theheterologous DNA sequence is operatively linked to an amplifiablemarker, in this example, the glutamine synthetase gene for which cellscan be amplified using methionine sulphoximine.

The pMDTM and pMSTOP vectors made in Example 2 were transfected into CHOcells as discussed below, according to the procedures of Crockett,Bio/Technology 8:662(1990) and as described in the Celltech InstructionManual for the glutamine synthetase gene amplification system (1992).

CHO-K1 cells (ATCC CCR61) were maintained in glutamine-free EMEM (EaglesMinimal Essential Medium) supplemented with 10% fetal bovine serum, 100units/ml penicillin, 100 mg/ml streptomycin, MEM (Modified Eagle'sMedium) nonessential amino acids, and 1 mM sodium pyruvate (all obtainedfrom JRH Biosciences). The media was also supplemented with 60 mg/mlglutamic acid, 60 mg/ml asparagine, 7 mg/ml adenosine, 7 mg/mlguanosine, 7 mg/ml cytidine, 7 mg/ml uridine, and 2.4 mg/ml thymidine(all from Sigma.) This media preparation was used throughout thetransfection, with deviations from this recipe as noted.

One day prior to transfection 10-cm dishes were seeded with 3×10⁶ CHO-K1cells. On the day of transfection the cells were washed with 10 mlserum-free media per dish. Plasmid DNA (from the pMDTM, pMSTOP plasmids)was applied by CaPO₄ precipitation using conventional techniques. 10 μgsof each plasmid DNA precipitate was incubated with the CHO-K1 cells plus2 ml of serum-free media at 37° C. for 4.5 hours. Three replicates ofeach of the four plasmid DNA transfections were made. The cells werethen shocked for 1.5 minutes with 15% glycerol in HEPES-buffered saline.After rinsing with serum-free media, the cells were re-fed withserum-containing media and incubated for 24 hours.

The following day the media was changed to include 10% dialyzed fetalbovine serum (JRH Biosciences) and amplified by the addition of 25 μMmethionine sulphoximine (Sigma). Cells were re-fed with methioninesulphoximine-containing media every 3-5 days until the amplified cloneswere large enough for picking, approximately 13-14 days later. Cloneswere picked by scraping colonies off the dish with a sterile 200 μlpipetman tip and transferred to one well of a 96-well plate in mediawithout methionine sulphoximine. 1-2 days later the media was replacedwith media+25 μM methionine sulphoximine. After 4 days the culturesupernatants were harvested and assayed for protein products in an ELISAassay, as discussed below.

CHO cells were also transfected with the pEE14 control vector alone(which contains no EBV sequences) and 24 clones of CHO-pEE14 were alsopicked and transferred to plates to serve as controls. (The controlclones were identified on the basis of survival in methioninesulphoximine.)

2. ELISA Assay

Following transfection, 241 clones of CHO-pMDTM and 158 clones ofCHO-pMSTOP were picked and grown up. Supernatants from these clones weretested for gp350 protein production. 96-well plates were coated withaffinity-purified rabbit anti-gp350/220 antibody (antibody MDP1; gift ofAndrew Morgan) diluted 1:2000 in 5 mM sodium borate buffer, pH 9. Theplates were incubated at 37° C. for 3-4 hours and washed 3 times withPBS+0.05% Tween 20 using a Nunc ImmunoWasher. After blotting dry, theplates were blocked by incubating with 2% BSA in PBS+0.01% Thimerosal at37° C. for 0.5 hours and washed again. Supernatants from the transfectedcells and control cells were added to the wells and incubated for 2hours at 37° C. The plates were then incubated with the primarydetection antibody, a mouse monoclonal antibody against gp350/220(antibody #C65221M; Biodesign International) at 1 mg/ml diluted in PBSwash buffer, 37° C. for 1 hour. After washing, the plates were incubatedwith the secondary antibody, horseradish peroxidase-conjugated goatF(ab)₂ fragments directed against mouse immunoglobulins (Human Igadsorbed; Biosource International.), 0.7 μg/ml in PBS+0.05% BSA and0.01% Thimerosal, at 37° C. for 1 hour. The plates were washed anddeveloped using ABTS (Pierce Chemicals) dissolved in Stable PeroxideSubstrate Buffer (Pierce Chemicals) for 0.5 hours at room temperature.The reaction was stopped with 1% SDS and the plates were read at 405 and650 nm wavelengths using a Molecular Devices Vmax ELISA plate reader. 24pMDTM and 18 pMSTOP clones tested positive for secreted gp350. Theclones exhibiting the highest ELISA signal were transferred to 24-wellplates for scale-up and further testing in a Western Blot and aradioimmunoprecipitation assay.

3. Western Blot and Radio Immunoprecipitation Assay

In an initial screening, tissue culture supernatants from the pMDTMtransfections were assayed for activity in a Western Blot. CHO cellsupernatants were purified on 5% SDS-PAGE gels, transferred tonitrocellulose overnight, and probed with anti-gp350 antibodies. SevenpMDTM clones were found to be positive for gp350 in the Western blotanalysis.

The pMDTM clones that were positive in the Western blot were furthertested by radioimmunoprecipitation for the presence of gp220. Selectedtransformed pMDTM cells, pEE14 control and GH3Δ19 control cells(described below) were grown overnight in six-well plates so that theywere approximately three-quarters confluent on the day of theexperiment. Each well contained approximately 5×10⁶ cells. Forlabelling, the media was removed from each well and replaced with 0.7 mlof methionine-free MEM (10% fetal calf serum)+100 μCi ³⁵S-methionine.The cells were incubated 5.5 hours at 37° C. and then microcentrifugedat 4000 rpm for 5 minutes. Homogeneous gp350 protein in the supernatantwas immunoprecipitated by addition of 10 μl of Sepharose-Protein A(Sigma) in a 50% slurry and 20 μl monoclonal anti-gp350/220 (antibody#C65221M, 100 mg/ml; Biodesign International), with overnight rocking at4° C. The mixture was then pelleted at 2000 rpm, 2 minutes at roomtemperature in a microcentrifuge and washed four times with severalvolumes of phosphate-buffered saline. After the final wash, all liquidwas removed from the pellet and replaced with 50 μl protein gel samplebuffer. The samples containing the precipitated immuno-complex wereboiled 5 minutes and run on a 5% SDS-PAGE. Immunoprecipitates werecompared to gel samples of tissue culture supernatants mixed 1:1 withprotein sample buffer. The gel was dried and autoradiographed withHyperfilm β-Max (Amersham).

FIG. 3 shows the autoradiographic results of SDS-PAGE analysis of theradioimmunoprecipitation. The cell line used as a positive control wasGH3Δ19 (gift of Elliot Keiff; Whang et al., 1987). GH3Δ19 cells secretea truncated form of the gp350/220 protein lacking the transmembrane andC-terminal cytoplasmic domains. For use as a negative control, CHO cellswere transfected with the pEE14 vector alone and selected by methioninesulphoximine in parallel with the pMDTM transfection. In FIG. 3,supernatants (“S”) are shown in odd numbered lanes, alternated withimmunoprecipitates (“Ip”) shown in even numbered lanes. In control lane2, precipitation from the GH3Δ19 control cells results in two strongprotein bands at approximately 220 and 350 kD demonstrating productionof the truncated splice variant gp350 and gp220 proteins in about a 1:1ratio. As expected, these immunoprecipitated bands are concentrated withrespect to the radiolabelled tissue culture supernatant(non-immunoprecipitated sample) in lane 1. Also, as expected, no bandsare shown in the negative control (lane 4), since the pEE14 vector doesnot contain any of the gp350/220 constructs.

SDS-PAGE analysis of the immunoprecipitation from supernatants of pMDTMclones in lanes 6, 8 and 10 results in a single strong band atapproximately 350 kD, the same as the higher molecular weight species inthe GH3Δ19 control lane 2. In contrast to the GH3Δ19 control lanehowever, an additional strong band at approximately 220 kD is absentfrom lanes 6, 8 and 10, although in lane 8 a very faint band migratingat a slightly lower molecular weight is revealed. This could represent adegradation product, a co-precipitated cellular product or a smallamount of gp220 protein resulting from a mistranslation or a mutationalevent that returns the deleted donor and acceptor splice sites to thenative nucleotide or amino acid sequences. Strong single bands atapproximately 350 kD were found in five other MTDM replicates tested(data not shown).

It is unlikely that the complete absence of the band at 220 kD in lanes6 and 10 is due to inefficient precipitation from MDTM supernatantssince in the ⁻S-labelled GH3Δ19 control lane (2), a band at 220 kD iseasily visualized. Also, additional assays using the pDTM constructs ofExample 1 that contain the wild type splice sites result in two strongbands at 350 and 220 kD. Therefore, these results demonstrate thatdeletion of the splice sites results in production of gp350 protein inthe absence of production of gp220 protein.

This homogeneous gp350 protein, expressed in CHO cell lines, or in othermammalian or non-mammalian cell lines, can be further scaled up andhomogenous gp350 protein can be isolated and purified from conditionedmedium from the cell line using methods familiar in the art, includingtechniques such as lectin-affinity chromatography, reverse phase HPLC,FPLC, gel filtration and the like. See David, J. Immunol. Methods108:231(1988) and Madej, Vaccine 10:777(1992).

EXAMPLE 4 Testing the Homogeneous gp350 Proteins for ImmunogenicActivity

The purified homogeneous gp350 proteins are incorporated intoappropriate vehicles for administration and administered to mice asfollows.

A 2×adjuvant-vehicle concentrate is prepared by mixing Pluronic L121 andsqualane in 0.4% (v/v) Tween 80 in phosphate buffered saline with (Thr¹)MDP in accordance with the procedure of David, J. Immunol. Methods108:231(1988) and Allison, J. Immunol. Methods 95:157(1986).

The composition for administration is prepared by addition of equalvolumes of protein and adjuvant-vehicle on the day of administration.The protein content should be with range of 5 micrograms to 50micrograms per dose.

BALB/c mice are immunized with three 0.1 ml intramuscular injections at0.21 and 42 days. A pre-immunization bleed and successive bleeds taken10 days after each injection are obtained from the retro-orbital sinus.

Serum antibody levels are determined by an ELISA according to theprocedures described in Example 3. EBV neutralizing antibodies in thesera are quantified by their ability to inhibit transformation of fetalcord blood lymphocytes by EBV in vitro according to the methods of Moss,J. Gen. Virol. 17:233(1972) and De Schryver, Int. J. Cancer13:353(1974).

Alternatively, New Zealand white rabbits are inoculated by intramuscularadministration of five doses of protein emulsified in the foregoingadjuvant at 0, 21, 42, 63 and 84 days. The dose should be in the rangeof about 5 μg to 50 μg per inoculation. Sera is obtained two weeksfollowing the last dose and tested for antibody titers to the antigen,for cross-reactive antibody to viral gp350/220 from B95-8 cells and forin vitro EBV-neutralizing activity following the methods of Emini,Virology 166:387(1988).

Because the ability of the EBV gp350/220 protein to induce protectiveimmunity in an animal model of EBV infection has already beenestablished, see Epstein, Clin. Exp. Immunol 63:485(1986), similarpositive results from administration of a homogeneous gp350 proteincomposition are expected.

The disclosures of all publication identified herein are expresslyincorporated herein by reference. The foregoing detailed description isgiven for clearness of understanding only and no unnecessary limitationsare either understood or inferred therefrom, as modifications within thescope of the invention will be obvious to those skilled in the art.

19 1 5931 DNA Virus gp350/220 1 gaattccata aatgaaacac gctggtcaggtgttaaaact tcctcccaga ttttcgtgag 60 gctcctgtgt atagccatat agtcaaagaaaatactgtag cggggattac agctctgtac 120 aatgttaccc acggagctct gaacatacaaccactggcga tccccggggg tacatcgcgg 180 cagcttaaag gtgccggcgg aaaaggtcacgtgacaccta cggccacctg tgcacccaag 240 tgtcgcctgg agatgtacga atgtgggagtcgtctggtga tcggtgtagc tgtacatcca 300 gctgctgtat gcctggtaac ccataggccatccggcggcc agggtttgca gtctccattt 360 ggcctgatct ctacgagaag ctggatttctccgacgatct ctaatggcct gtcgaatggc 420 catggcatac attatgtaca tctcggtatttgaaatctgg atccgaaaaa ctggtctatg 480 gctcgtgtgt cgatgcgctg aaaccaacggcaacaaatta cttaccttgt tgttgtgtga 540 tgggtaaaaa cacacatcac acacttaggccatagggatg ctcaccgtag ccgcggctcc 600 aatcgcttga agaagtgttc ttagatctagtggaaacctg cggagaatgg cttctcgccc 660 agggagatcc ggctggggtg ggagcatgggtcgtgctgga gctgacccac cggcatcatg 720 atcgacccgc tttctcttcg tacccttctgggccggctcc aggtgggcat cttctgcttc 780 cttttctgag ctgctatctg ataactctatgaggacattt tcccaatctc ccgccgatac 840 ctgttcctgc acaaccgagg tagatgggacttcttcttcc atgttgtcat ccagggccgg 900 gggacccggc ctgtccttgt ccattttgtctgcaacaaaa gtgtgactct ccaacaccgc 960 accccccttg tacctattaa agaggatgctgcctagaaat cggtgccgag acaatggagg 1020 cagccttgct tgtgtgtcag tacaccatccagagcctgat ccatctcacg ggtgaagatc 1080 ctggtttttt caatgttgag attccggaattcccatttta ccccacatgc aatgtttgca 1140 cggcagatgt caatgtaact atcaatttcgatgtcggggg caaaaagcat caacttgatc 1200 ttgactttgg ccagctgaca ccccatacgaaggctgtcta ccaacctcga ggtgcatttg 1260 gtggctcaga aaatgccacc aatctctttctactggagct ccttggtgca ggagaattgg 1320 ctctaactat gcggtctaag aagcttccaattaacgtcac caccggagag gagcaacaag 1380 taagcctgga atctgtagat gtctactttcaagatgtgtt tggaaccatg tggtgccacc 1440 atgcagaaat gcaaaacccc gtgtacctgataccagaaac agtgccatac ataaagtggg 1500 ataactgtaa ttctaccaat ataacggcagtagtgagggc acaggggctg gatgtcacgc 1560 tacccttaag tttgccaacg tcagctcaagactcgaattt cagcgtaaaa acagaaatgc 1620 tcggtaatga gatagatatt gagtgtattatggaggatgg cgaaatttca caagttctgc 1680 ccggagacaa caaatttaac atcacctgcagtggatacga gagccatgtt cccagcggcg 1740 gaattctcac atcaacgagt cccgtggccaccccaatacc tggtacaggg tatgcataca 1800 gcctgcgtct gacaccacgt ccagtgtcacgatttcttgg caataacagt atcctgtacg 1860 tgttttactc tgggaatgga ccgaaggcgagcgggggaga ttactgcatt cagtccaaca 1920 ttgtgttctc tgatgagatt ccagcttcacaggacatgcc gacaaacacc acagacatca 1980 catatgtggg tgacaatgct acctattcagtgccaatggt cacttctgag gacgcaaact 2040 cgccaaatgt tacagtgact gccttttgggcctggccaaa caacactgaa actgacttta 2100 agtgcaaatg gactctcacc tcggggacaccttcgggttg tgaaaatatt tctggtgcat 2160 ttgcgagcaa tcggacattt gacattactgtctcgggtct tggcacggcc cccaagacac 2220 tcattatcac acgaacggct accaatgccaccacaacaac ccacaaggtt atattctcca 2280 aggcacccga gagcaccacc acctcccctaccttgaatac aactggattt gctgatccca 2340 atacaacgac aggtctaccc agctctactcacgtgcctac caacctcacc gcacctgcaa 2400 gcacaggccc cactgtatcc accgcggatgtcaccagccc aacaccagcc ggcacaacgt 2460 caggcgcatc accggtgaca ccaagtccatctccatggga caacggcaca gaaagtaagg 2520 cccccgacat gaccagctcc acctcaccagtgactacccc aaccccaaat gccaccagcc 2580 ccaccccagc agtgactacc ccaaccccaaatgccaccag ccccacccca gcagtgacta 2640 ccccaacccc aaatgccacc agccccaccttgggaaaaac aagtcctacc tcagcagtga 2700 ctaccccaac cccaaatgcc accagccccaccttgggaaa aacaagcccc acctcagcag 2760 tgactacccc aaccccaaat gccaccagccccaccttggg aaaaacaagc cccacctcag 2820 cagtgactac cccaacccca aatgccaccggccctactgt gggagaaaca agtccacagg 2880 caaatgccac caaccacacc ttaggaggaacaagtcccac cccagtagtt accagccaac 2940 caaaaaatgc aaccagtgct gttaccacaggccaacataa cataacttca agttcaacct 3000 cttccatgtc actgagaccc agttcaaacccagagacact cagcccctcc accagtgaca 3060 attcaacgtc acatatgcct ttactaacctccgctcaccc aacaggtggt gaaaatataa 3120 cacaggtgac accagcctct atcagcacacatcatgtgtc caccagttcg ccagaacccc 3180 gcccaggcac caccagccaa gcgtcaggccctggaaacag ttccacatcc acaaaaccgg 3240 gggaggttaa tgtcaccaaa ggcacgcccccccaaaatgc aacgtcgccc caggccccca 3300 gtggccaaaa gacggcggtt cccacggtcacctcaacagg tggaaaggcc aattctacca 3360 ccggtggaaa gcacaccaca ggacatggagcccggacaag tacagagccc accacagatt 3420 acggcggtga ttcaactacg ccaagaccgagatacaatgc gaccacctat ctacctccca 3480 gcacttctag caaactgcgg ccccgctggacttttacgag cccaccggtt accacagccc 3540 aagccaccgt gccagtcccg ccaacgtcccagcccagatt ctcaaacctc tccatgctag 3600 tactgcagtg ggcctctctg gctgtgctgacccttctgct gctgctggtc atggcggact 3660 gcgcctttag gcgtaacttg tctacatcccatacctacac caccccacca tatgatgacg 3720 ccgagaccta tgtattaaag tcaataaaaatttattaatc agaatttgca ctttctttgc 3780 ttcacgtccc cgggagcggg agcgggcacgtcgggtggcg ttggggtcgt ttgattctcg 3840 tggtcgtgtt ccctcaccag ggctgggttggccttttgca cccaacatag atacttgaat 3900 gcggagggtc agattttgca atatattttccatttcattg cgggtagtta caccgtcaac 3960 agatttccga accttgtctt caatcttcttcatagcctga gacgccaaac ggcccgtggc 4020 ctgcgtaatc attacctctc gctgtcgagctgtaagcggc tcaggcggag gcactggagc 4080 agaaggaaca gaggtagacg aggcacaggcaccccctctg aggacctgtt gcttcagagc 4140 cttattctca gactccaggc gagccaggcgggcggccatg tcttccatgc tcatgtcaac 4200 agccttaaca gaaggcaatc tgactttgcgtggagctgac atgctattgg tttaacgagc 4260 agagaagaag tacaaacagc cgagattgctgcccctttta aatatcccca tctaatccgt 4320 cagcagcgtg ttcacaaact tgttaaagcagacgtacatc aggtagatgg ctgaggctat 4380 aatgactaag acaagcgtca gaagtgcccagatggaggca aagctggtca aagaaggcga 4440 gaatgtgtcc gcattacatg tgtaggagtagaactggtcc tgggcttcag tcagggaagc 4500 cgccgccgcg tttgtgggac tggaagcgccggccggcagc accccggtca gaagccaggt 4560 ggcggcgagg aagagcagtg gcacagcctttgcaaacggc tttcttagga ccttccccat 4620 ttcgcagtaa agagagccgg gtcttgggctcttatatata gcgccgccgt ccctgtctgt 4680 tagatcatca ccatggaggc ctgtccacacatacgctacg ccttccagaa tgacaagctg 4740 ttgctccagc aggagctcaa aatccacctggctgccttca gatatgccac ccccctgctg 4800 cgcccgatga agaccacaac tgtggacctaggcctctatg cccgcccacc cgagggtcat 4860 gggctcatgc tgtggggcag cacctcccgtccggtcacgt ctcatgttgg catcatcgat 4920 cccggctaca cgggggaact ccggctaatcctccagaatc agcggcgcta caactccacg 4980 ctgcgtccat cggagctcaa aatccacctggctgccttca gatatgccac cccccagatg 5040 gaggaggaca agggtcccat caaccacccccagtaccccg gggacgtggg cctggacgtc 5100 tctttgccaa aggacctggc cctcttcccccatcagaccg tctcagtgac actcaccgtg 5160 cccccccctt ctatccctca ccacaggccgacaatctttg gcaggtcggg cctggccatg 5220 cagggtattc tagtgaagcc ctgcaggtggcgccggggtg gggtggacgt cagcctgacc 5280 aactttagtg accagaccgt gttccttaacaagtaccggc gcttctgtca gcttgtttac 5340 cttcacaagc accacctcac ctccttctacagcccccaca gtgacgcggg ggtccttggc 5400 cccagatctc tctttaggtg ggccagctgcaccttcgagg aggtgccgag cctggccatg 5460 ggtgatagtg ggctgagcga ggcgctcgaggggagacagg ggagggggtt tggatcctcg 5520 ggtcaatgac accttcatat cccttgttttaccaataaaa tgtttatttg gtgtggagtc 5580 tggttgctac gttaacgcga gctccgtgggcccagagtgc tccggctgcc gcaccacggg 5640 aggcggtgca aggacggggg tgggcacctcgggctcaaag gcggagttga tgaaaggggc 5700 cgaggctgag ggggcggtca ccgatgagacaacggccgag atctcttgcg cccaggcacc 5760 ggtctcgtcc atcttgagcg ccgtggccaccttctccatc tccatcaggg ccaggctgtc 5820 gccagcctgt ttgtccagca gggccttgagcccattctcg tccatctcga tgccgcttac 5880 agccagagag atcatggtat tccagatgactgagcgcacg gcctcaagct t 5931 2 878 PRT Virus gp350 2 Met Glu Ala Ala LeuLeu Val Cys Gln Tyr Thr Ile Gln Ser Leu Ile 1 5 10 15 His Leu Thr GlyGlu Asp Pro Gly Phe Phe Asn Val Glu Ile Pro Glu 20 25 30 Phe Pro Phe TyrPro Thr Cys Asn Val Cys Thr Ala Asp Val Asn Val 35 40 45 Thr Ile Asn PheAsp Val Gly Gly Lys Lys His Gln Leu Asp Leu Asp 50 55 60 Phe Gly Gln LeuThr Pro His Thr Lys Ala Val Tyr Gln Pro Arg Gly 65 70 75 80 Ala Phe GlyGly Ser Glu Asn Ala Thr Asn Leu Phe Leu Leu Glu Leu 85 90 95 Leu Gly AlaGly Glu Leu Ala Leu Thr Met Arg Ser Lys Lys Leu Pro 100 105 110 Ile AsnVal Thr Thr Gly Glu Glu Gln Gln Val Ser Leu Glu Ser Val 115 120 125 AspVal Tyr Phe Gln Asp Val Phe Gly Thr Met Trp Cys His His Ala 130 135 140Glu Met Gln Asn Pro Val Tyr Leu Ile Pro Glu Thr Val Pro Tyr Ile 145 150155 160 Lys Trp Asp Asn Cys Asn Ser Thr Asn Ile Thr Ala Val Val Arg Ala165 170 175 Gln Gly Leu Asp Val Thr Leu Pro Leu Ser Leu Pro Thr Ser AlaGln 180 185 190 Asp Ser Asn Phe Ser Val Lys Thr Glu Met Leu Gly Asn GluIle Asp 195 200 205 Ile Glu Cys Ile Met Glu Asp Gly Glu Ile Ser Gln ValLeu Pro Gly 210 215 220 Asp Asn Lys Phe Asn Ile Thr Cys Ser Gly Tyr GluSer His Val Pro 225 230 235 240 Ser Gly Gly Ile Leu Thr Ser Thr Ser ProVal Ala Thr Pro Ile Pro 245 250 255 Gly Thr Gly Tyr Ala Tyr Ser Leu ArgLeu Thr Pro Arg Pro Val Ser 260 265 270 Arg Phe Leu Gly Asn Asn Ser IleLeu Tyr Val Phe Tyr Ser Gly Asn 275 280 285 Gly Pro Lys Ala Ser Gly GlyAsp Tyr Cys Ile Gln Ser Asn Ile Val 290 295 300 Phe Ser Asp Glu Ile ProAla Ser Gln Asp Met Pro Thr Asn Thr Thr 305 310 315 320 Asp Ile Thr TyrVal Gly Asp Asn Ala Thr Tyr Ser Val Pro Met Val 325 330 335 Thr Ser GluAsp Ala Asn Ser Pro Asn Val Thr Val Thr Ala Phe Trp 340 345 350 Ala TrpPro Asn Asn Thr Glu Thr Asp Phe Lys Cys Lys Trp Thr Leu 355 360 365 ThrSer Gly Thr Pro Ser Gly Cys Glu Asn Ile Ser Gly Ala Phe Ala 370 375 380Ser Asn Arg Thr Phe Asp Ile Thr Val Ser Gly Leu Gly Thr Ala Pro 385 390395 400 Lys Thr Leu Ile Ile Thr Arg Thr Ala Thr Asn Ala Thr Thr Thr Thr405 410 415 His Lys Val Ile Phe Ser Lys Ala Pro Glu Ser Thr Thr Thr SerPro 420 425 430 Thr Leu Asn Thr Thr Gly Phe Ala Asp Pro Asn Thr Thr ThrGly Leu 435 440 445 Pro Ser Ser Thr His Val Pro Thr Asn Leu Thr Ala ProAla Ser Thr 450 455 460 Gly Pro Thr Val Ser Thr Ala Asp Val Thr Ser ProThr Pro Ala Gly 465 470 475 480 Thr Thr Ser Gly Ala Ser Pro Val Thr ProSer Pro Ser Pro Trp Asp 485 490 495 Asn Gly Thr Glu Ser Lys Ala Pro AspMet Thr Ser Ser Thr Ser Pro 500 505 510 Val Thr Thr Pro Thr Pro Asn AlaThr Ser Pro Thr Pro Ala Val Thr 515 520 525 Thr Pro Thr Pro Asn Ala ThrSer Pro Thr Pro Ala Val Thr Thr Pro 530 535 540 Thr Pro Asn Ala Thr SerPro Thr Leu Gly Lys Thr Ser Pro Thr Ser 545 550 555 560 Ala Val Thr ThrPro Thr Pro Asn Ala Thr Ser Pro Thr Leu Gly Lys 565 570 575 Thr Ser ProThr Ser Ala Val Thr Thr Pro Thr Pro Asn Ala Thr Ser 580 585 590 Pro ThrLeu Gly Lys Thr Ser Pro Thr Ser Ala Val Thr Thr Pro Thr 595 600 605 ProAsn Ala Thr Gly Pro Thr Val Gly Glu Thr Ser Pro Gln Ala Asn 610 615 620Ala Thr Asn His Thr Leu Gly Gly Thr Ser Pro Thr Pro Val Val Thr 625 630635 640 Ser Gln Pro Lys Asn Ala Thr Ser Ala Val Thr Thr Gly Gln His Asn645 650 655 Arg Pro Ser Ser Asn Pro Glu Thr Leu Ser Pro Ser Thr Ser AspAsn 660 665 670 Ser Thr Ser His Met Gly Gly Glu Asn Ile Thr Gln Val ThrPro Ala 675 680 685 Ser Ile Ser Thr His His Val Ser Thr Ser Ser Pro GluPro Arg Pro 690 695 700 Gly Thr Thr Ser Gln Ala Ser Gly Pro Gly Asn SerSer Thr Ser Thr 705 710 715 720 Lys Pro Gly Glu Val Asn Val Thr Lys GlyThr Pro Pro Gln Asn Ala 725 730 735 Thr Ser Pro Gln Ala Pro Ser Gly GlnLys Thr Ala Val Pro Thr Val 740 745 750 Thr Ser Thr Gly Gly Lys Ala AsnSer Thr Thr Gly Gly Lys His Thr 755 760 765 Thr Gly His Gly Ala Arg ThrSer Thr Glu Pro Thr Thr Asp Tyr Gly 770 775 780 Gly Asp Ser Thr Thr ProArg Pro Arg Tyr Asn Ala Thr Thr Tyr Leu 785 790 795 800 Pro Pro Ser ThrSer Ser Lys Leu Arg Pro Arg Trp Thr Phe Thr Ser 805 810 815 Pro Pro ValThr Thr Ala Gln Ala Thr Val Pro Val Pro Pro Thr Ser 820 825 830 Gln ProArg Phe Ser Asn Leu Ser Met Leu Val Leu Leu Leu Leu Leu 835 840 845 LeuVal Met Ala Asp Cys Ala Phe Arg Arg Asn Leu Ser Thr Ser His 850 855 860Thr Tyr Thr Thr Pro Pro Tyr Asp Asp Ala Glu Thr Tyr Val 865 870 875 3 27DNA Artificial Sequence primer 3 ggatcctaga ctgcgccttt aggcgta 27 4 19DNA Artificial Sequence primer 4 gactgcgcct ttaggcgta 19 5 6 PRTArtificial Sequence peptide 5 Asp Cys Ala Phe Arg Arg 1 5 6 27 DNAArtificial Sequence primer 6 ggatcctctg ttccttctgc tccagtg 27 7 21 DNAArtificial Sequence primer 7 tctgttcctt ctgctccagt g 21 8 16 DNAArtificial Sequence primer 8 tatagactag tctagg 16 9 18 DNA ArtificialSequence primer 9 atctgatcag atccttaa 18 10 39 DNA Artificial Sequenceprimer 10 ccgcgtcagg cggtactggt catgatcgta cctctccaa 39 11 13 PRT Virus11 Ala Cys Asp Ala Met Val Leu Val Leu Met Ser Leu Asn 1 5 10 12 30 DNAVirus 12 ggatcttgat cagatatcgt acctctccaa 30 13 5 PRT ArtificialSequence peptide 13 Leu Met Ser Leu Asn 1 5 14 24 DNA ArtificialSequence primer 14 ccgcgtcaga tcgtacctct ccaa 24 15 8 PRT ArtificialSequence peptide 15 Ala Cys Asp Leu Met Ser Leu Asn 1 5 16 42 DNAArtificial Sequence Mutagenesis oligonucleotide as PrDonor1 16ggtcatgtcg ggggcctttg actctgtgcc gttgtcccat gg 42 17 42 DNA Virus 17ggtcatgtcg ggggccttac tttctgtgcc gttgtcccat gg 42 18 42 DNA ArtificialSequence Mutagenesis oligonucleotide as PrAcceptor1 18 ctgtgttatattttcacctc cagttgggtg agcggaggtt ag 42 19 42 DNA Virus 19 ctgtgttatattttcaccac ctgttgggtg agcggaggtt ag 42

We claim:
 1. A method of producing a gp350 protein for use inpharmaceutical compositions comprising: (a) culturing host cellstransformed with a DNA sequence encoding EBV gp350 protein having adeletion in the membrane spanning region and the remaining carboxyterminus, and/or a deletion of the signal sequence, said DNA sequence,having a mutation at one or more splice sites preventing formation of agp220 mRNA transcript; and (b) isolating the expressed homogeneous gp350from the cell and culture medium.
 2. The method of claim 1 wherein thehomogeneous gp350 is further isolated by ultrafiltration, gelfiltration, ion exchange, and hydrophobic interaction chromatography. 3.The method of claim 1 or 2 wherein the homogeneous gp350 is formulatedin admixture with a pharmaceutically acceptable carrier.
 4. The methodof claim 3 wherein the pharmaceutically acceptable carrier is anadjuvant/antigen presentation system.
 5. The method of claim 1 whereinsaid mutation at one or more splice sites is a mutation in the donorsplice site.
 6. The method of claim 1 wherein said mutation at one ormore splice sites is a mutation in the acceptor splice site.
 7. Themethod of claim 5 or 6 in which at least one native nucleotide encodingserine at codon 501 of SEQ. ID. No.: 18 is replaced with a non-nativenucleotide, and in which at least one native nucleotide encoding glycineat codon 698 of SEQ. ID. No.: 18 is replaced with a non-nativenucleotide.
 8. The method of claim 6 wherein the DNA encodes a shortenedversion of EBV gp350 having a deletion of at least 8 amino acids in themembrane spanning region, resulting in a secreted product.
 9. The methodof claim 8 wherein said mutation at one or more splice sites is amutation in the donor splice site.
 10. The method of claim 8 whereinsaid mutation at one or more splice site is a mutation in the acceptorsplice site.